Mechanism of alkaline phosphatase

Figure 12.12 Principal steps in the mechanism of alkaline phosphatase. (Reprinted with permission from Parkin, 2004. Copyright (2004) American Chemical Society.)...

FIGURE 19. Reaction mechanism of alkaline phosphatase. For clarity, the complete active site is shown only in the first strucmre. Adapted with permission from Reference 10. Copyright (2005) ACS... 

Fig. 8 Catalytic mechanism of alkaline phosphatase reaction [44]. The initial alkaline phosphatase (E)-cataiyzed reaction consists of a substrate (DO-Pi) binding step, phosphate-moiety transfer to Ser-93 (in the TNAP sequence of its active site), and product alcohol (DOH) release. In the second part of the reaction, phosphate is released through hydrolysis of the covalent intermediate (E-Pi) and non-covaient compiex (E Pi) of inorganic phosphate in the active site. In the presence of alcohol molecules (AOH), phosphate is aiso reieased via a transphosphorylation reaction...

Liver injury is clinically defined as an increase of serum alanine amino transferase (ALT) levels of more than three times the upper limit of normal and a total bilirubin level of more than twice the upper limit of normal [4]. The clinical patterns of liver injury can be characterized as hepatocellular (with a predominant initial elevation of ALT), cholestatic (with an initial elevation of alkaline phosphatase) or mixed. The mechanisms of drug-induced hepatotoxicity include excessive generation of reactive metabolites, mitochondrial dysfunction, oxidative stress and inhibition of bile salt efflux protein [5]. Better understandings of these mechanisms in the past decades led to the development of assays and models suitable for studying such toxic mechanisms and for selecting better leads in the drug discovery stage. 

Table 2 Effects of alkaline phosphatase concentration on the gelation time and mechanical properties of the peptide hydrogels of Emoc-Y-OH (entries 1-4) and...

The mechanism by which the more recently used WR-2721 (Fig. 8) reduces nephrotoxicity is not very well understood. WR-2721 protects against nephrotoxicity when administered just prior to cis-Pt (144,145) and it is known that WR-2721 is preferentially taken up by normal cells and not by tumor cells (146). Recently, it was concluded that the uncharged form of the dephosphorylated WR-2721 (known as WE-1065) is the actual species taken up by both normal and tumor cells (147). It has been proposed that the conversion of WR-2721 to WR-1065 is slower in tumors, compared with normal tissues (147), possibly because tumors generally have lower levels of alkaline phosphatase (148). Furthermore, it has been proposed (147) that once formed, WR-1065 will have a decreased uptake rate in tumors, probably as a consequence of their lower pH (149) as compared with normal tissues i.e., the neutral form of WR-1065 will only constitute 0.1% of the total drug present at pH 7 and 1% of the total at pH 8. The reactive WR-1065 is likely to bind directly to cis-Pt, thereby preventing side reactions of cis-Pt. 

Although inversion was not observed with the E. colt alkaline phosphatase, it has been observed for ribonucleases and many other hydrolytic enzymes and for most kinases transferring phospho groups from ATP. The difference lies in the existence of a phospho-enzyme intermediate in the action of alkaline phosphatase (see Eq. 12-38). Each of the two phosphotransferase steps in the phosphatase action apparently occurs with inversion. The simplest interpretation of all the experimental results is that phosphotransferases usually act by in-line -like mechanisms which may involve metaphosphate-ion-like transition states that are constrained to react with an incoming nucleophile to give inversion. An adjacent attack with pseudorotation would probably retain the original configuration and is therefore excluded. 

More recently, isotopic labeling experiments have assumed a major role in establishing the detailed mechanism of enzymic action. It was shown that alkaline phosphatase possesses transferase activity whereby a phos-phoryl residue is transferred directly from a phosphate ester to an acceptor alcohol (18). Later it was found that the enzyme could be specifically labeled at a serine residue with 32P-Pi (19) and that 32P-phosphoserine could also be isolated after incubation with 32P-glucose 6-phosphate (20), providing strong evidence that a phosphoryl enzyme is an intermediate in the hydrolysis of phosphomonoesters. The metal-ion status of alkaline phosphatase is now reasonably well resolved (21-23). Like E. coli phosphatase it is a zinc metalloenzyme with 2-3 g-atom of Zn2+ per mole of enzyme. The metal is essential for catalytic activity and possibly also for maintenance of native enzyme structure. 

The transfer of phosphate is stimulated by l,25-(OH)2D3, but little is known about the mechanism. Vitamin D also stimulates the activities of alkaline phosphatase and Ca2+-ATPase. This may be involved in the supply of inorganic phosphate. 

Williams, A. and R. A. Naylor. 1971. Evidence foifoS(P) mechanism in the phosphorylation of alkaline phosphatase by substrate. Chem. Soc. B973-1979. 

Resistance to both 6-MP and 6-TG occurs most commonly by decrease in HGPRT activity an alternative mechanism in acute leukemia involves elevation of levels of alkaline phosphatase, which results in dephosphorylation of thiopurine nucleotide and cellular loss of the resulting ribonucleoside. 

The third example is a phosphoryl transfer enzyme, alkaline phosphatase. The active site of alkaline phosphatase contains two Zn + ions, with a separation of 3.9 A. One zinc center is used to bind the phosphate monoester substrate, the other to activate Ser-102 for nucleophilic attack on the phosphate group of the substrate via an associative mechanism, as shown... 

Schlaeger R, Hairs P, Kattermann R. Studies on the mechanism of the increase in serum alkaline phosphatase activity in cholestasis Significance of the hepatic bile acid concentration for the leakage of alkaline phosphatase from rat liver, En2yme 1982 28 3-13. 

Interesting indeed are the recent studies of Griffin and Cox (G20, G21) on the mechanism of adrenocorticoid induction of alkaline phosphatase in human cell cultures. Two temporal phases in the induction of enzyme in HeLa cells are reported a first 12-hour period exhibiting a slow rise in activity, followed by a sudden marked linear increase extending from 15 to 80 hours. The elevation of activity is marked (around 10-fold). The fact that puromycin blocked induction immediately, and actinomycin D... 

G20. Griffin, M. J., and Cox, R. P., Studies on the mechanism of hormonal induction of alkaline phosphatase in human cell cultures. I. Effects of Puromycin and Actinomycin D. J. Cell Biol. 29, 1-9 (1966). 

M6. Manson, L. A., Pelmont, J., Yapo, A., Roche, C., and Nisman, B., The biosynthesis of alkaline phosphatase with a particulate fraction of Escherichia coli. The mechanism of inhibition by inorganic phosphate. Biochem. J. 96, 215-225 (1965). 

Lomashvili KA, Garg P, Narisawa S et al (2008) Upregulation of alkaline phosphatase and pyrophosphate hydrolysis potential mechanism for luremic vascular calcification. Kidney Int 73 1024-1030... 

In some kinases, such as nucleoside diphosphate kinase, " an intermediate step is the phosphoryl transfer to a group belonging to the enzyme, as happens in ATPase and as was discussed in detail for alkaline phosphatase (Section V.B). In other kinases the phosphoryl transfer occurs directly from the donor to the acceptor in a ternary complex of the enzyme with the two substrates.Often metal ions like magnesium or manganese are needed. These ions interact with the terminal oxygen of the ATP molecule, thus facilitating the nucleophilic attack by the acceptor. The metal ion is often associated with the enzyme. For mechanistic schemes, see the proposed mechanism of action of alkaline phosphatase, especially when a phosphoryl enzyme intermediate is involved. 

Figure 30. Proposed mechanism of action of alkaline phosphatase.

The reader is referred to the following sources of further information on the specific cocatalytic enzymes superoxide dismutase, and the reduced form, alkaline phosphatases, nuclease PI, purple acid phosphatase, amidohydrolase, leucine aminopeptidase, general comments on the mechanisms of the phosphatases and aminopeptidases, and other cocatalytic zinc enzymes. ... 

Mark, S.S., Stolper, S.I., Baratti, C., Park, J.Y., Taku, M.A., Santiago-AvUes, J.J. and Kricka, L.J. 2008. Bioconjugation of alkaline phosphatase to mechanically processed, aqueous suspendible eiectrospun polymer nanofibers for use in chemiluminescent detection assays. Macromof igscf 8 484—498. 

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